Project Summary Persistent inflammation and immune activation drive HIV disease progression despite ART therapy. However, the underlying mechanisms are currently speculative and not clearly delineated. Gut leakiness and microbial translocation are hallmarks of HIV disease progression. Interestingly, chronic opioid abuse is also well documented to induce gut leakiness and sustained microbial translocation. Increasing number of studies strongly support the concept that the gut microbiota, play a significant role in maintaining gut homeostasis and gut barrier function. Although a few studies have correlated the host microbiome in HIV infected patients with gut barrier function disruption and microbial translocation, the consequence in the context of antiretroviral treatment (ART) still remains largely unexplored. There is no data on the consequences of an altered microbiome with immune activation and viral persistence in HIV patients on ART therapy in the context of opioid abuse. Although most studies measure endotoxin levels and bacterial products associated with gram- negative bacterial infection with inflammation and HIV disease progression, recent studies clearly show a distinct enrichment and prevalence of gram positive bacterial communities in HIV infected patients when compared to normal healthy individuals. Our preliminary data show significant expansion and translocation of gram positive bacterial communities in HIV infected BLT mice which are further exacerbated in the context of opioid treatment. Our central hypothesis is that microbial dysbiosis with preferential expansion of Gram positive (G+) bacterial communities in HIV infected individuals and HIV infected individuals that are opioid abusers lead to G+ bacterial translocation, TLR2/TLR4 activation and is the driving mechanism for immune cell activation and sustained inflammation contributing to disease progression and preventing the restoration of normal health in HIV-infected individuals that are opioid drug abusers. We will further investigate if treatment with ART restores homeostasis or exacerbates dysbiosis. We will test our hypothesis using NSG-BLT humanized murine model of HIV and HIV in context of substance abuse and ART. In Specific Aim 1 we will determine the role of microbial dysbiosis, expansion of gram-positive bacterial communities and gut leakiness with immune activation and viral persistence. In Specific Aim 2 we will determine that activation of TLR-2 in immune cells and gut epithelial cells results in immune activation and viral persistence following HIV infection and in the context of opioid drug abuse and ART treated humanized mice. In Specific Aim 3 we will determine that combination therapy with Probiotics and TLR2 antagonists will restore gut homeostasis and attenuate immune activation thereby delaying HIV disease progression in the context of opioid abuse and ART. The results from these studies will allow for the development of new adjunct therapeutic strategies to attenuate immune activation and delay HIV disease progression both in HIV infected patients that are on ART and in HIV infected drug abusing population.